The present invention relates to methods for the preparation of 3xe2x80x2-O and 5xe2x80x2-O-levulinyl nucleosides from common precursors using an enzymatic approach. These methods are useful for the large-scale synthesis of oligonucleotides.
It is well known that most of the bodily states in mammals, including most disease states, are affected by proteins. Such proteins, either acting directly or through their enzymatic functions, contribute in major proportion to many diseases in animals and man. Classical therapeutics has generally focused on interactions with such proteins in efforts to moderate their disease causing or disease potentiating functions. Recently, however, attempts have been made to moderate the actual production of such proteins by interactions with molecules that direct their synthesis, such as intracellular RNA. By interfering with the production of proteins, it has been hoped to affect therapeutic results with maximum effect and minimal side effects. It is the general object of such therapeutic approaches to interfere with or otherwise modulate gene expression leading to undesired protein formation.
One method for inhibiting specific gene expression is the use of oligonucleotides and oligonucleotide analogs as xe2x80x9cantisensexe2x80x9d agents. The oligonucleotides or oligonucleotide analogs complimentary to a specific, target, messenger RNA (mRNA) sequence are used. Antisense methodology is often directed to the complementary hybridization of relatively short oligonucleotides and oligonucleotide analogs to single-stranded mRNA or single-stranded DNA such that the normal, essential functions of these intracellular nucleic acids are disrupted. Hybridization is the sequence specific hydrogen bonding of oligonucleotides or oligonucleotide analogs to Watson-Crick base pairs of RNA or single-stranded DNA. Such base pairs are said to be complementary to one another.
Oligonucleotides and oligonucleotide analogs are now accepted as therapeutic agents holding great promise for therapeutics and diagnostics methods. But applications of oligonucleotides and oligonucleotide analogs as antisense agents for therapeutic purposes, diagnostic purposes, and research reagents often require that the oligonucleotides or oligonucleotide analogs be synthesized in large quantities.
Three principal methods have been used for the synthesis of oligonucleotides. The phosphotriester method, as described by Reese, Tetrahedron 1978, 34, 3143; the phosphoramidite method, as described by Beauage, in Methods in Molecular Biology: Protocols for Oligonucleotides and Analogs; Agrawal, ed.; Humana Press: Totowa, 1993, Vol. 20, 33-61; and the H-phosphonate method, as described by Froehler in Methods in Molecular Biology: Protocols for Oligonucleotides and Analogs Agrawal, ed.; Humana Press: Totowa, 1993, Vol. 20, 63-80.
The phosphotriester approach has been widely used for solution phase synthesis, whereas the phosphoramidite and H-phophonate strategies have found application mainly in solid phase syntheses. Recently, Reese reported a new approach to the solution phase synthesis of oligonucleotides on H-phosphonate coupling. See, Reese et al. Nucleic Acids Research, 1999, 27, 963-971, and Reese et al. Biorg. Med. Chem. Lett. 1997, 7, 2787-2792, which is incorporated herein by reference. Solution phase synthesis is the method of choice in producing large-scale quantities of oligonucleotides.
These solution phase methods require the use of nucleoside monomer building blocks bearing protecting groups on the 3xe2x80x2-O and/or the 5xe2x80x2-O positions. The protecting groups should be stable to coupling conditions and selectively cleaved without affecting other protecting groups in the molecule. One such protecting group is the levulinyl group, xe2x80x94C(O)xe2x80x94(CH2)2xe2x80x94C(O)xe2x80x94CH3. However, the preparation of nucleosides bearing these protecting groups involves several tedious chemical protection/deprotection and/or purification steps.
For example, the 3xe2x80x2,5xe2x80x2-di-O-levulinyl protection of nucleosides can be accomplished using a well-established method wherein nucleosides are selectively acylated at their hydroxyl sites by reacting the nucleosides with levulinic acid in the presence of DCC (dicyclohexylcarbodiimide). Despite the utility of this method, it suffers from at least one significant problem. The method requires a large excess of DCC to achieve optimal yields. The excess DCC is converted to DCU (dicyclohexylcarbodiimide) upon completion of the reaction, which must be separated from the reaction mixture. Unfortunately, for large-scale syntheses, the separation step requires considerable time and expense.
Prior to the present invention, synthesis of 5xe2x80x2-O-levulinyl nucleosides was accomplished by reacting parent nucleosides with levulinic acid and 2-chloro-1-methylpyridinium iodide. Iwai et al., Nucleic Acids Res. 1988, 16, 9443-9456; Iwai et al. Tetrahedron 1990, 46, 6673-6688. However, because this method does not afford selective acyaltion of the 5xe2x80x2-hydroxyl function, additional purification and deprotection steps are necessary because both 3xe2x80x2-acyl and 3xe2x80x2,5xe2x80x2-diacyl derivatives are formed in the reaction. After the 3xe2x80x2,5xe2x80x2-diacyl derivatives are separated by chromatography, the residue must be treated with DMTrCl to remove the 3xe2x80x2-acyl compound. Finally, an additional purification by chromatography isolates the 5xe2x80x2-O-levulinyl derivatives in very low yields.
Before now, the synthesis of 3xe2x80x2-O-levulinyl nucleosides (2xe2x80x2-deoxy or 2xe2x80x2-protected) was accomplished by the treatment of parent nucleosides with levulinic acid or levulinic anhydride and DCC. One of the major drawbacks of this method is that it requires that the 5xe2x80x2-hydroxyl function be protected as a 5xe2x80x2-O-DMTr group prior to acylation with levulinic acid. The 5xe2x80x2-O-DMTr group must then be removed in an acid medium to afford the 3xe2x80x2-O- protected nucleosides. See, Reese et al., Nucleic Acids Res. 1999, 27, 963-971, and Reese et al., J. Chem. Soc., Perkin Trans. 1 1999, 1477-1486.
Commercially viable methods for the large-scale synthesis of oligonucleotides are constantly being explored. It has been found that the application of biocatalysts in organic synthesis has become an attractive alternative to conventional chemical methods. See, Carrea, et al. Angew. Chem. Int. Ed. 2000, 39, 2226-2254; Bornscheuer, et al. Hydrolases in Organic Synthesis. Regio- and Stereoselective Biotransformations; Wiley-VCH: Weinheim, 1999. Enzymes catalyze reactions with high chemo-, regio-, and stereoselectivity. See, Ferrero et al. Chem. Rev. 2000, 100, 4319-4347; Ferrero et al., Monatsh. Chem. 2000, 131, 585-616. It has previously been reported that Candida antarctica lipase B (CAL-B) catalyzes acylation at the 5xe2x80x2-hydroxyl group of nucleosides with high selectivity. Pseudomonas cepacia lipase (PSL) shows unusual regioselectivity towards the secondary alcohol at the 3xe2x80x2-position of 2xe2x80x2-deoxynucleosides. Moris et al., J. Org. Chem. 1993, 58, 653-660; Gotor et al. Synthesis 1992, 626-628.
In the last few years the use of antisense oligonucleotides has emerged as an exciting new therapeutic paradigm. As a result, very large quantities of therapeutically useful oligonucleotides are required in the near future. In view of the considerable expense and time required for synthesis of oligonucleotide building blocks, there has been a longstanding effort to develop successful methodologies for the preparation of oligonucleotides with increased efficiency and product purity.
Applicants have discovered methods that are useful in, for example, the large-scale synthesis of oligonucleotides. The methods of the present invention help to minimize the number of steps required to yield desired results using an enzymatic approach. Applicants have found that both 3xe2x80x2-O-levulinyl nucleosides and 5xe2x80x2-O-levulinyl nucleosides can be prepared from a common precursor the regioselective deprotection of a 3xe2x80x2, 5xe2x80x2-di-O-levulinyl nucleoside to yield the desired 3xe2x80x2-O-levulinyl nucleoside or 5xe2x80x2-O-levulinyl nucleoside. Surprisingly, it has been found that the presence of selected lipases in deprotection reaction protocols gives rise to regioselectivity of deprotection
According to one embodiment, a method is provided for regioselectively deprotecting a 3xe2x80x2,5xe2x80x2-di-O-levulinylnucleoside comprising selecting a lipase that is effective to direct a regioselective hydrolysis of one of the levulinyl positions, without causing an undesired level of hydrolysis on the other of the levulinyl positions, and contacting the 3xe2x80x2,5xe2x80x2-di-O-levulinyl nucleoside with the lipase for a time and under conditions effective to yield either a 3xe2x80x2-O-levulinyl or a 5xe2x80x2-O-levulinyl nucleoside. Examples of lipases that are amenable to the present invention include Candida antarctica lipase B (CAL-B), Candida antarctica lipase A (CAL-A), Pseudomonas cepacia lipase (PSL), porcine pancreatic lipase, Chromobacteriaum viscosum lipase, Mucor miehei lipase, Humicola lanuginosa lipase, Penicillium camemberti lipase, Candida rugosa lipase, and others.
According to an embodiment of the present invention, a 3xe2x80x2,5xe2x80x2-di-O-levulinyl nucleoside is deprotected at the 5xe2x80x2-O-levulinyl position by contacting the diprotected nucleoside with CAL-B for a time and under conditions effective to regioselectively hydrolyze the 5xe2x80x2-O-levulinyl position without affecting the 3xe2x80x2-O-levulinyl position.
In another embodiment, a 3xe2x80x2-, 5xe2x80x2-di-O-levulinyl nucleoside is deprotected at the 3xe2x80x2-O levulinyl position by contacting the diprotected nucleoside with CAL-A or PSL-C for a time and under conditions effective to regioselectively hydrolyze the 3xe2x80x2-O-levulinyl position without affecting the 5xe2x80x2-O-levulinyl position.
In some embodiments of the present invention, methods are disclosed for regioselectively deprotecting a 3xe2x80x2-, 5xe2x80x2-di-O-levulinyl nucleoside at the 5xe2x80x2-O-levulinyl position wherein the nucleoside has one of the following formulas: 
wherein:
R1 is xe2x80x94H, -hydroxyl, a protected hydroxyl, a 2xe2x80x2-substituent or a 2xe2x80x2-protected substituent; and
R2 and R3 are, independently, xe2x80x94H or an amino protecting group;
G is N or CH; and
Lev is xe2x80x94C(O)xe2x80x94(CH2)2xe2x80x94C(O)xe2x80x94CH3, the levulinyl group;
comprising selecting a lipase that is effective to direct a regioselective hydrolysis of the 5xe2x80x2-O-levulinyl position, without causing hydrolysis on the 3xe2x80x2-O-levulinyl position, and contacting the 3xe2x80x2,5xe2x80x2-di-O-levulinyl nucleoside with the lipase for a time and under conditions effective to yield a 3xe2x80x2-O-levulinyl nucleoside. A preferred lipase for 5xe2x80x2-O-levulinyl hydrolysis is CAL-B.
In still further embodiments, methods are provided for regioselectively deprotecting a nucleoside at the 3xe2x80x2-O-levulinyl position wherein the nucleoside has one of the following formulas: 
wherein:
R6 is xe2x80x94H, -hydroxyl;
R2, R3, R4, and R5 are each, independently, xe2x80x94H or an amino protecting group;
G is N or CH; and
Lev is xe2x80x94C(O)xe2x80x94(CH2)2xe2x80x94C(O)xe2x80x94CH3;
comprising selecting a lipase that is effective to direct a regioselective hydrolysis of the 3xe2x80x2-O-levulinyl position, without causing hydrolysis of the 5xe2x80x2-O-levulinyl position, and contacting the 3xe2x80x2,5xe2x80x2-di-O-levulinyl nucleoside with the lipase for a time and under conditions effective to yield a 5xe2x80x2O-levulinyl nucleoside. Lipases that are preferable for hydrolysis at the 3xe2x80x2-O-levulinyl positions are, for example, CAL-A or PSL-C.
In some embodiments of the present invention, methods for acylating a hydroxyl moiety of a nucleic acid, such as a nucleoside or a nucleotide, at at least one of a 2xe2x80x2-O, 3xe2x80x2-O, or 5xe2x80x2-O position are provided comprising reacting the nucleic acid with levulinic acid in the presence of a coupling agent, such as a carbodiimide, that is attached to a polymeric support for a time and under conditions effective to form an ester at the 2xe2x80x2-O, 3xe2x80x2-O or 5xe2x80x2-O position. Preferred polymeric supports comprise polystyrene or polyethylene glycol polymeric supports that are attached to cyclohexylcarbodiimide.
The present invention includes the esterification or acylation of any hydroxyl moiety, such as those found in carbohydrates or steroid molecules, by reacting the compounds containing the hydroxyl moiety with levulinic acid in the presence of a coupling agent that is attached to a polymeric support for a time and under conditions effective to form an ester between the hydroxyl moieties and the levulinyl group of the levulinic acid. In some embodiments of the present invention, methods are provided for acylating at least one hydroxyl moiety on a compound having the following formula: 
wherein:
Bx is a nucleobase;
T1 and T2 are, independently, -hydroxyl, a hydroxyl protecting group, an activated phosphate group, a nucleotide, a nucleoside, or an oligonucleotide;
R is xe2x80x94H, -hydroxyl, a protected hydroxyl or a 2xe2x80x2 substituent group;
provided that at least one of T1, T2 or R is -hydroxyl;
comprising reacting the compound with levulinic acid in the presence of a coupling agent that is attached to a solid support, such as PS-cyclohexylcarbodiimide, for a time and under conditions effective to form an ester between the hydroxyl moiety and the levulinyl group. In a preferred embodiment, T1 and T2 are xe2x80x94OH and R is xe2x80x94H or a 2xe2x80x2-substituent.
In one preferred embodiment, methods are provided for acylating the 3xe2x80x2-O and 5xe2x80x2-O positions of a compound having the following formula: 
wherein:
Bx is a nucleobase; and
R is hydroxyl or an optionally protected 2xe2x80x2-substituent comprising reacting the compound with levulinic acid in the presence of a coupling agent that is attached to a solid support for a time and under conditions effective to form a compound having formula: 
wherein Lev is -levulinyl.
According to one embodiment of the present invention, methods are provided for generating a cyclohexylcarbodiimide derivatized polymeric support from a cyclohexylurea derivatized polymeric support comprising reacting the cyclohexylurea derivatized polymeric support with a dehydrating agent, such as tosyl chloride or POCl3, in an organic solvent for a time and under conditions effective to yield the cyclohexylcarbodiimide derivatized polymeric support. In some embodiments, the organic solvent employed is CH2Cl2, CHCl3, hexane, or pyridine.
In a further embodiment of the present invention, a method is provided for generating a cyclohexylcarbodiimide derivatized polymeric support from a cyclohexylurea derivatized polymeric support comprising the steps of reacting the cyclohexylurea derivatized polymer support with a dehydrating agent for a time and under conditions effective to form a salt and subsequently contacting the salt with an aqueous solution, such as aqueous NaOH, to form the cyclohexylcarbodiimide derivatized polymeric support.